INTRODUCTION
The most used drugs on the treatment of parasitic infections are benzimidazoles such as albendazole, fenbendazole, oxfendazole, mebendazole and tricyclabendazole. These drugs affect both intestinal and tissue parasites due to their mode of action which is the blockage of microtubule and tubulin formation within the parasite's cell (Barot et al. Reference Barot, Nikolova, Ivanov and Ghate2013). This blockage results in several biochemical effects within the parasite which contribute to its death, such as glucose uptake impairment, induction of alternative energetic pathways and consequent death by starvation (Vinaud et al. Reference Vinaud, Lino Junior and Bezerra2007; Fraga et al. Reference Fraga, Costa, Bezerra, Lino Junior and Vinaud2012a ; Márquez-Navarro et al. Reference Márquez-Navarro, Pérez-Reyes, Zepeda-Rodrigues, Reynoso-Ducoing, Hernández-Campos, Hernández-Luis, Castillo, Yépez-Muli and Ambrosio2013). Few benzimidazole derivatives have been studied with regard to their biochemical effect, for instance, albendazole, cambendazole, oxibendazole and thiabendazole dissipate the transmembrane proton gradient resulting in diminished ATP levels (McCracken and Stillwell, Reference McCracken and Stillwell1991). The biochemical studies of benzimidazole derivatives may help to understand their mode of action and to determine their specificity within the metabolic pathways that are essential for the parasite's survival (McCracken and Stillwell, Reference McCracken and Stillwell1991; Feldmeier, Reference Feldmeier2010; Geary, Reference Geary2012).
Worldwide Taeniidae parasites still remain important public health issues which make them the target of several studies such as biochemical, physiological, susceptibility to drugs and others (Hoberg et al. Reference Hoberg, Jones, Rausch, Emon and Gardner2000). Due to their strikingly high incidence both in humans and in animals these parasites are important indicators of health and sanitary conditions of the population (Aragão et al. Reference Aragão, Biondi, Lima and Nunes2010; Coral-Almeida et al. Reference Coral-Almeida, Gabriël, Abatih, Praet, Benitez and Dorny2015; Scala et al. Reference Scala, Pipia, Dore, Sanna, Tamponi, Marrosu, Bandino, Carmona, Boufana and Varcasia2015).
The most common drugs used against helminthes are albendazole and praziquantel. Their mode of action is related to tegument damage, calcium channels impairment and inhibit of β-tubulin polimerization. These effects lead to metabolic effects such as glucose uptake impairment and the induction of anaerobic metabolism and alternative pathways for energy production (Venkatesan, Reference Venkatesan1998; Vinaud et al. Reference Vinaud, Lino Junior and Bezerra2007, Reference Vinaud, Ferreira, Lino Junior and Bezerra2008, Reference Vinaud, Ferreira, Lino Junior and Bezerra2009).
Albendazole has been widely used as anti-helminthic treatment against intestinal and tissue parasites (Feldmeier, Reference Feldmeier2010; Del Brutto, Reference Del Brutto2014). However, due to its use for longer than 20 years, albendazole resistance reports have been made (Márquez-Navarro et al. Reference Márquez-Navarro, Cornejo-Coria, del, Cebada-López, Sánchez-Manzano, Díaz-Chiguer and Nogueda-Torres2012; Lopes et al. Reference Lopes, Cruz, Soares, Nunes, Teixeira, Maciel, Buzzulini, Pereira, Felippelli, Soccol, de Oliveira and da Costa2014). Therefore studies aiming to improve the efficiency of the albendazole molecule and its mode of action have been conducted (Márquez-Navarro et al. Reference Márquez-Navarro, Pérez-Reyes, Zepeda-Rodrigues, Reynoso-Ducoing, Hernández-Campos, Hernández-Luis, Castillo, Yépez-Muli and Ambrosio2013; Matadamas-Martínez et al. Reference Matadamas-Martínez, Nogueda-Torres, Hernández-Campos, Hernández-Luis, Castillo, Mendoza, Ambrosio, Andrés-Antonio and Yépez-Mulia2013; Garćia et al. Reference García, Leonardi, Salazar and Lamas2014). One albendazole derivative has evidenced promising activity – the 6-chloro-5-(1-naphthyloxy)-2-(trifluoromethyl)-1H-benzimidazole (RCB20). This derivative presented a better in vitro activity on Taenia crassiceps cysticerci than sulfoxide albendazole (ABZSO) regarding morphological parameters (Márquez-Navarro et al. Reference Márquez-Navarro, Pérez-Reyes, Zepeda-Rodrigues, Reynoso-Ducoing, Hernández-Campos, Hernández-Luis, Castillo, Yépez-Muli and Ambrosio2013).
The fatty acids oxidation, ketone body production and urea cycle are metabolic pathways described in T. crassiceps and other cestodes used for energy production and essential for the parasite's survival. The parasite removes from the environment or from reserve vacuoles the substrates for these pathways with considerable ATP production. In situations of environmental stress such as the presence of antihelminthic drugs that impair the glucose uptake the parasite enhances these metabolic pathways enabling its survival (Vinaud et al. Reference Vinaud, Ferreira, Lino Junior and Bezerra2009; Fraga et al. Reference Fraga, Costa, Bezerra, Lino Junior and Vinaud2012b ). Therefore the studies of the metabolic range of the parasites when exposed to drugs may indicate the pathways used to escape metabolic distress and a possible resistance development (Barot et al. Reference Barot, Nikolova, Ivanov and Ghate2013).
As albendazole is capable of in vitro and in vivo enhancing the use of alternative pathways for energy production by T. crassiceps cysticerci (Vinaud et al. Reference Vinaud, Ferreira, Lino Junior and Bezerra2009; Fraga et al. Reference Fraga, Costa, Bezerra, Lino Junior and Vinaud2012a ), this study aimed the evaluation of the fatty acid oxidation and urea cycle in T. crassiceps cysticerci in vitro exposed to RCB20, considering that it is a molecule similar to albendazole with different chemical properties.
MATERIALS AND METHODS
Maintenance of T. crassiceps cysticerci
The maintenance of T. crassiceps cysticerci was performed according to the description by Fraga et al. (Reference Fraga, Costa, Bezerra, Lino Junior and Vinaud2012a ). The ethical principles of animal experimentation stipulated by the Brazilian Society of Laboratory Animals Science (SBCAL) were obeyed. This study was approved by the Ethics in Research Committee from the Federal University of Goias (CoEp/UFG) (protocol number 011/11).
Cysticerci culture and drugs exposure
A mouse with 30 days of intraperitoneal infection was euthanized within a laminar flow chamber and the cysticerci were removed and washed with physiological solution as to remove cells and any other contaminants (Márquez-Navarro et al. Reference Márquez-Navarro, Pérez-Reyes, Zepeda-Rodrigues, Reynoso-Ducoing, Hernández-Campos, Hernández-Luis, Castillo, Yépez-Muli and Ambrosio2013). Afterwards the cysticerci were macroscopically classified into its evolutive stages: initial (no buds, vesicular membrane and fluid translucent), larval (with buds, vesicular membrane and fluid translucent) and final (no buds, vesicular membrane and fluid opaque) (Vinaud et al. Reference Vinaud, Lino Junior and Bezerra2007).
The cysticerci culture was performed as described by Vinaud et al. (Reference Vinaud, Ferreira, Lino Junior and Bezerra2008) and Márquez-Navarro et al. (Reference Márquez-Navarro, Pérez-Reyes, Zepeda-Rodrigues, Reynoso-Ducoing, Hernández-Campos, Hernández-Luis, Castillo, Yépez-Muli and Ambrosio2013). Briefly, 30 cysticerci from each evolutive stage were added into 5 mL of supplemented RPMI culture medium. The treatments were as follows: albendazole sulfoxide (ABZSO) (Sigma-Aldrich®) (6·5 and 13 µ m) and RCB20 (6·5 and 13 µ m). The control groups of each evolutive stage did not receive the drugs. There were other control groups of each evolutive stage that received dimethyl sulfoxide (DMSO) at the same concentration used to dilute the drugs.
After 24 h of culture the cysticerci were separated from their culture medium and frozen into liquid nitrogen as to stop the metabolic reactions (Vinaud et al. Reference Vinaud, Ferreira, Lino Junior and Bezerra2008, Reference Vinaud, Ferreira, Lino Junior and Bezerra2009).
Biochemical analysis
The organic acids secreted/excreted into the culture medium were extracted through an ionic exchange solid phase extraction column (Bond Elut® Agilent®), as described by Vinaud et al. (Reference Vinaud, Lino Junior and Bezerra2007).
After the liquid nitrogen metabolic stasis, the cysticerci were defrost and homogenized in 500 µL of tris–HCl 0·1 m buffer supplemented with a protease inhibitor (SIGMAFAST, protease inhibitor cocktail tablets, EDTA free, Sigma), pH 7·6 (Rendón et al. Reference Rendón, Del Arenal, Guevara-Flores, Uribe and Mendoza-Hernández2004, Reference Rendón, Del Arenal, Guevara-Flores, Mendonza-Hernández and Pardo2008). The extract obtained was centrifuged at 15 652 g (10 000 rpm) per 10 min at 4 °C and then the organic acids were extracted through an ionic exchange solid phase extraction column (Bond Elut® Agilent®) (Vinaud et al. Reference Vinaud, Lino Junior and Bezerra2007).
The resulting samples were frozen at −20 °C for posterior analysis in high-performance liquid chromatography.
For the chromatographic analysis an exclusion BIORAD-Aminex HPX-87H column was used. The eluent was sulphuric acid 5 mm, 0·6 mL min−1, with spectrophotometric reading of absorbance at 210 nm. The results were analysed through the Star Chromatography Workstation software (Agilent®), previously calibrated for the following organic acids identification: acetate, acetoacetate, β-hydroxybutyrate, propionate and fumarate (Vinaud et al. Reference Vinaud, Lino Junior and Bezerra2007). The analysis of the proteins catabolism was performed through the quantification of urea, creatinin and total proteins in the culture medium which was performed through an Architec C8000 Plus device, using a commercial kit protocol and enzymatic method (Fraga et al. Reference Fraga, Costa, Bezerra, Lino Junior and Vinaud2012b ).
Statistical analysis
All experiments were repeated five times independently. The statistical analysis was performed through the Sigma Stat 2·3 software. The descriptive analysis was performed as to determine the normal distribution and homogenous variation as well as mean and standard deviation. As the values presented normal distribution, the analysis-of-variation test (ANOVA) was performed. The differences were considered significant when P < 0·05.
RESULTS
This study showed the metabolic effect of two benzimidazole derivatives (RCB20 and ABZSO) in a widely used experimental model, T. crassiceps cysticerci. It was possible to evaluate both the quantification of organic acids within the cysticerci and in the culture medium (secreted/excreted by the parasite) in three different evolutive stages of the parasite, initial, larval and final ones. It was possible to detect the fatty acid oxidation and the protein catabolism in all cysticerci stages through the quantification of acetate, acetoacetate, β-hydroxybutyrate, propionate, fumarate, urea, creatinin and total proteins (Tables 1 and 2). Both RCB20 and ABZSO were diluted in DMSO, which did not interfere in the parasite's metabolic pathways.
Mean concentrations (μ m) of organic acids of the fatty acid oxidation and volatile fatty acids detected in the medium culture (secreted/excreted) and in the cysticerci of Taenia crassiceps in vitro exposed to ABZSO and RCB20. Results expressed in mean ± standard deviation
CM, culture medium; Cy, cysticerci; ND, non-detected; RCB20, 6-chloro-5-(1-naphtyloxy)-2-(trifluorometil)-1H-benzimidazol; ABZSO, albendazole sulfoxide.
* Statistical difference when compared to the concentrations detected in the culture medium (t test, P < 0·05).
Mean concentrations of fumarate (μ m), urea (mg dL−1), creatinin (mg dL−1) and total proteins (g dL−1) in the culture medium of Taenia crassiceps cysticerci in vitro exposed to ABZSO and RCB20. Results expressed in mean ± standard deviation
RCB20, 6-chloro-5-(1-naphtyloxy)-2-(trifluorometil)-1H-benzimidazol; ABZSO, albendazole sulphoxide; Cy, cysticerci; CM, culture medium.
a Statistical difference when compared with the concentrations detected in the culture medium (t test).
Bold and *: statistical difference when compared with the control group (ANOVA).
Fatty acid oxidation
The acetate concentrations were detected mostly in the culture medium and not within the cysticerci. The detection of this organic acid in the cysticerci occurred only in initial stages ones with RCB20 6·5 and ABZSO 13 µ m.
It was not possible to detect acetoacetate in the control group, i.e., which did not receive any treatment, neither in the cysticerci nor in the culture medium. However in the treated groups, this organic acid was detected in the cysticerci from all stages (treated with ABZSO and RCB20 at 6·5 µ m), initial stage (treated with RCB20 13 µ m) and final stage (treated with ABZSO 13 µ m).
The β-hydroxybutyrate detection occurred mainly in the culture medium analysis of the initial and larval stages cysticerci from the control group and the ones treated with both drugs at 6·5 µ m. In the final stages cysticerci, it was not possible to detect this organic acid neither in the culture medium nor in the cysticerci with the exception of the group treated with RCB20 13 µ m which presented a higher concentration in the culture medium than in the cysticerci. In the other treated groups, when this organic acid was detected its concentration was higher in the cysticerci than in the culture medium.
The propionate detection occurred mainly in the culture medium with the exception of initial stages cysticerci both from the control group and treated ones. The propionate concentrations detected in the cysticerci analysis of the initial stage ones treated with both concentrations of ABZSO were significantly higher than in the culture medium (P < 0·05). In the larval and final stage cysticerci treated with RCB20 13 µ m, propionate was not detected neither in the cysticerci nor in the culture medium.
Proteins catabolism
It was only possible to analyse the creatinin, urea and total proteins concentrations on the culture medium. All cysticerci analysed presented a functional urea cycle which was detected through the quantification of fumarate, creatinin, urea and total proteins. There was no significant difference in the creatinin and total proteins quantification. On the other hand, the urea concentrations detected in the culture medium were increased in final stage cysticerci treated with ABZSO 6·5 µ m when compared with the same evolutive stage in the control group. The RCB20 treatment did not influence the proteins catabolism. The fumarate concentrations were higher in all the analysis of the cysticerci when compared with the culture medium (P < 0·05) (Table 2).
DISCUSSION
This study evaluated the in vitro metabolic effect of two benzimidazole derivatives on the alternative energetic pathways of T. crassiceps cysticerci, an experimental model widely used for metabolic analysis of Taeniidae (Vinaud et al. Reference Vinaud, Lino Junior and Bezerra2007, Reference Vinaud, Ferreira, Lino Junior and Bezerra2008, Reference Vinaud, Ferreira, Lino Junior and Bezerra2009; Fraga et al. Reference Fraga, Costa, Bezerra, Lino Junior and Vinaud2012a , Reference Fraga, Costa, Bezerra, Lino Junior and Vinaud b ; Márquez-Navarro et al. Reference Márquez-Navarro, Pérez-Reyes, Zepeda-Rodrigues, Reynoso-Ducoing, Hernández-Campos, Hernández-Luis, Castillo, Yépez-Muli and Ambrosio2013). The need of benzimidazole derivatives have been explored in the literature with promising results (Barot et al. Reference Barot, Nikolova, Ivanov and Ghate2013) due to the extensive use of albendazole and the appearance of resistance cases, especially within veterinary use (Geary, Reference Geary2012; Márquez-Navarro et al. Reference Márquez-Navarro, Cornejo-Coria, del, Cebada-López, Sánchez-Manzano, Díaz-Chiguer and Nogueda-Torres2012).
The acetoacetate detection occurred mainly in the cysticerci of the treated groups because it is the β-hydroxybutyrate precursor (Tielens et al. Reference Tielens, van Grinsven, Henze, van Hellemond and Martin2010) and also because in those groups probably a greater fatty acid oxidation occurred. Also in some groups (control, RCB20 13 µ m) it was not possible to detect this organic acid, but the β-hydroxybutyrate detection occurred. This happened due to the consumption of acetoacetate to β-hydroxybutyrate production. On the other hand, the treatment with 6·5 µ m concentrations of both RCB20 and ABZSO induced a greater production of this organic acid in which the β-hydroxybutyrate production was also increased both in the culture medium and in the cysticerci analyses. Probably this happened due to a greater oxidation of fatty acids in the cysticerci exposed to those concentrations. This metabolic response in T. crassiceps cysticerci in vivo and in vitro exposed to albendazole was also described by Vinaud et al. (Reference Vinaud, Ferreira, Lino Junior and Bezerra2009) and Fraga et al. (Reference Fraga, Costa, Bezerra, Lino Junior and Vinaud2012b ). Also the higher detection of β-hydroxybutyrate in the cysticerci of the treated groups when compared with the control ones may happen due to the mode of action of RCB20 and albendazole which alters the tubulin formation impairing the secretion mechanisms of the cells (Márquez-Navarro et al. Reference Márquez-Navarro, Pérez-Reyes, Zepeda-Rodrigues, Reynoso-Ducoing, Hernández-Campos, Hernández-Luis, Castillo, Yépez-Muli and Ambrosio2013). Therefore the whole secretion mechanism of the parasite is affected. On the other hand, acetate and propionate which are volatile fatty acids do not need tubulins to be carried out of the cytoplasm (Ahring et al. Reference Ahring, Sandberg and Angelidaki1995) and that is why they were mainly detected in the culture medium analysis (secreted/excreted by the parasite).
Also as propionate is the end product of the succinate fermentation via succinyl-, methylmalonyl- and propionyl-coenzime A (Reichardt et al. Reference Reichardt, Duncan, Young, Belenger, Leitch, Scott, Flint and Louis2014), this pathway indicates energy production by the parasite which is increased in initial stage cysticerci treated with ABZSO 6·5 and 13 µ m.
The fumarate detection was higher in the cysticerci than in the culture medium as it is an organic acid which participates both in the urea cycle and in the tricarboxilic acid cycle. Therefore its secretion/excretion occurs only when it is in excess. As the parasites were in the culture medium with great amounts of glucose, amino acids and fatty acids all the energetic pathways were available to them (Tielens et al. Reference Tielens, van Grinsven, Henze, van Hellemond and Martin2010). Also the drugs, in the concentrations tested in this study, did not interfere in the urea cycle.
The urea cycle was also detected in T. crassiceps cysticerci as an energy production pathway from the proteins catabolism. Such cycle has been described previously in this parasite by Vinaud et al. (Reference Vinaud, Ferreira, Lino Junior and Bezerra2009) in in vitro studies and by Fraga et al. (Reference Fraga, Costa, Bezerra, Lino Junior and Vinaud2012a ) in in vivo studies. It is important to highlight that the parasite is capable of performing simultaneously several energetic rentable pathways such as glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, the urea cycle, the fatty acids oxidation, among others (Barret, Reference Barret2009; Tielens et al. Reference Tielens, van Grinsven, Henze, van Hellemond and Martin2010). In addition when it is metabolic challenged, both in vivo and in vitro, some of these pathways are enhanced or decreased (Vinaud et al. Reference Vinaud, Lino Junior and Bezerra2007, Reference Vinaud, Ferreira, Lino Junior and Bezerra2008, Reference Vinaud, Ferreira, Lino Junior and Bezerra2009; Fraga et al. Reference Fraga, Costa, Bezerra, Lino Junior and Vinaud2012a , Reference Fraga, Costa, Bezerra, Lino Junior and Vinaud b ).
The concentrations of ABZSO and RCB20 used in this study were the ones described by Márquez-Navarro et al. (Reference Márquez-Navarro, Pérez-Reyes, Zepeda-Rodrigues, Reynoso-Ducoing, Hernández-Campos, Hernández-Luis, Castillo, Yépez-Muli and Ambrosio2013) in which structural effects were observed in in vitro T. crassiceps cysticerci. In spite of the morphological alterations and the different expression of tubulin isoforms these concentrations do not cause great biochemical alterations in the metabolic pathways, only in the secretion/excretion mechanism. Further studies with higher concentrations in in vivo analysis should be performed as to confirm RCB20 as an alternative benzimidazolic drug.
It is possible to infer that the structural alteration in the RCB20 molecule when compared with the ABZSO one does not interfere in its metabolic effect acting as a promising alternative to albendazole as the biochemical effects occur in the same targets. Therefore we conclude that the metabolic effects on alternative energetic pathways are slightly increased in the parasites treated with RCB20 than the ones treated with ABZSO.
FINANCIAL SUPPORT
J.A. received financial support from the DGAPA-UNAM (grant no. IN-216213) and M.C.V. received financial support from the CNPQ (grant no. 471009/2013-0).
